1. Field of the Invention
The present invention includes methods for treating conditions modulated by lactosylceramide and, more particularly, to the use of one or more compounds that enhance UDP-galactose, GlcCer, xcex21xe2x86x924 galactosyltransferase (GalT-2) activity to treat a subject suffering from or susceptible to a condition caused or contributed to by lactosylceramide. The present invention also relates to methods for detecting and analyzing compounds with therapeutic capacity to treat such conditions.
2. Background
Inappropriate cell proliferation in an organism can modulate the development or severity of a variety of conditions. For example, there is recognition that certain conditions can be treated or prevented by increasing cell proliferation in adult or pre-adult animals. In particular, it has been proposed that conditions relating to infection, ulceration, degeneration (e.g., apoptotic and necrotic cell death), aging, hematopoiesis, angiogenesis, certain immune responses, cell and tissue repair can be positively impacted by increasing the proliferation of specified cells. See generally Alberts, B. et al. (1989) in Molecular Biology of the Cell 2nd ed. Garland Publishing Co. (New York and London); Kandel, E. R et al. (1991) in Principles of Neuroscience, Apppleton and Lange, (Norwalk, Conn.); Cold Spring Harbor Conf. Cell Proliferation (1979), Cold Spring Harbor Laboratories, (New York); Tissue Growth Factors, (1981) R. Baseega, ed., (Springer-Verlag, New York).
The proliferation of particular animal cells has attracted interest. For example, the proliferation of smooth-muscle cells (SMCs), epithelia, and other intima has been reported to effect vascular development and integrity, e.g., as in vascular malformation and formation of vascular lesions. In addition, the proliferation of certain skin cells is believed to enhance responses to various traumata such as wounding (e.g., following thermal injury). It is particularly recognized that SMCs and epithelia have significant roles in angiogenesis. See e.g., Tissue Growth Factors, supra; Folkman and Shing (1992), J. Biol. Chem. 267: 10931.
The proliferation of cells associated with heart, brain, liver, kidney, eye and other organs has also attracted attention. For example, it has been suggested that increasing numbers of specified cells can treat or prevent certain neurodegenerative diseases such as those impacting the central and peripheral (e.g., motor) systems. Degenerative diseases of the retina and other eye structures can lead to progressive deterioration of vision. In particular, age-related macular dystrophies (e.g., Stargardt disease) are believed to be negatively impacted by inappropriate proliferation of certain cells, e.g., macula. It is has been proposed that the effect of many, if not all neurodegenerative disorders can be offset by enhancing the proliferation of specified cells. See e.g., Kusiak, J. W et al (1996) Mol. Chem. Neuropathol. 153; Kandel, E. R et al. supra; Neary et al. (1996) Trends Neurosci. (1996) 13.
There has been recognition that inappropriate cell adhesion can also contribute to some conditions. For example, it has been suggested that blood coagulation is enhanced by adhesion of platelets and perhaps other blood cells to injured vessels. In addition, certain immune responses, e.g., inflammation associated with rejection of foreign bodies, and recruitment of immune cells are believed to be enhanced in many cases by cell adhesion. Increased adhesion of certain cells may also augment angiogenesis following trauma, during development or following grafting.
A variety of synthetic, semi-synthetic and naturally-occurring cell molecules have been reported to play significant roles in animal cell proliferation. Such molecules include certain cytokines, growth factors, cell receptors, matrix molecules, enzymes, second messenger molecules (e.g., cyclic nucleotides) transcription factors, and mitogens such as phorbol esters.
Other molecules such as adhesion molecules appear to have significant roles in initiating and maintaining suitable cell-to-cell contact.
More particularly, molecules with capacity to modulate cell pathways comprising glycosphingolipids (GSLs).have attracted substantial interest. The GSLs have been reported to have roles in the proliferation and adhesion of animal cells among other functions. See e.g., Chatterjee, S., Biochem. Biophys. Res Comm. (1991) 181:554; Hakomori, S. I. (1983) in Sphingolipid Chemistry, eds. Kanfer, J. N. and Hakomori, S. I. (Plenum Press, New York); Obeid, L. M et al. (1993) Science 259: 1769 and references cited therein.
Specific cell pathways relating to GSLs such as glucosylceramide (GlcCer) and lactosylceramide (LacCer) have been disclosed. For example, one pathway involves synthesis of GlcCer by coupling UDP-glucose to ceramide in a reaction catalyzed by UDP-glucose glucosyltransferase (GlcT-1). Another step converts the GlcCer to LacCer using UDP-galactose, GlcCer, xcex21xe2x86x924 galactosyltransferase (GalT-2). See e.g., Chatterjee et al. supra.
Attempts have been made to inhibit the steps involving GlcT-1. For example, it has been reported that the D-enantiomer of 1-phenyl-2-decanolylamino-3-morpholino-1-propanol (D-PDMP) inhibits GlcT-1 and reduces proliferation of vascular cells. The mechanism of PDMP action has been reported to be unclear. See e.g., Felding-Habermann, B., et al. (1991) Biochemistry 29:6314; Shukla, G. S. et al. Biochem. Biophys. Acta. (1991) 1083:101; Inokuchi, J. et al., J. Lipid. Res. (1987) 28:565; and Chatterjee, S., supra.
Specified morpholinoceramides also have been disclosed as GlcT-1 inhibitors. See Carson, K. and B. Ganem (1994) Tetrahedron Lets. 35:2659.
Increased levels of LacCer are believed to enhance the proliferation of certain animal cells such as aortic smooth-muscle cells and specified melanoma cells. See e.g., Chatterjee, S., supra and Noirijiri, H. Et al (1988) J. Biol. Chem. 263:443.
Thus, it would be desirable to have effective methods of modulating levels of LacCer e.g., by enhancing GalT-2 activity. In particular, it would be useful to have therapeutic methods of increasing LacCer levels to treat or prevent conditions or diseases impacted by lactosylceramides.
We have now discovered therapies to treat or prevent various conditions or diseases modulated by lactosylceramide (LacCer). In particular, we have discovered therapies that include increasing activity of UDP-galactose, GlcCer, xcex21xe2x86x924 galactosyl-transferase (GalT-2).
More specifically, the invention provides methods for treatment or prevention of conditions or diseases impacted by increased cell proliferation or adhesion, e.g., tissue repair, ulceration, blood coagulation, infection, degeneration (e.g., apoptotic and necrotic cell death), angiogenesis, aging and certain immune responses and chemoattraction.
Therapies of the invention are particularly effective for enhancing tissue repair and for the treatment or prevention of undesired degeneration particularly involving cells such as neurons of the central (CNS) or peripheral (PNS) nervous system including the eye. In one protocol of the invention, an increase in cell proliferation following cell or tissue contact with one or more compositions of the invention can be observed, whereas a control exhibits much less proliferation. In particular, the present invention features a variety of in vitro and in vivo protocols for testing the compositions as set forth in the discussion and examples which follow.
LacCer-modulated conditions that can be enhanced in accordance with the invention also include angiogenesis (neovascularization); and response to various traumata e.g., ulceration of smooth muscle and related cells; tissue repair, particularly in response to burning or an incision; and grafting.
Therapeutic methods of the invention in general comprise administering to a subject, particularly a mammal such as a primate and especially a human, in need of treatment a therapeutically effective amount of a compound that can promote GalT-2 activity. Preferably, an administered compound increases cell proliferation by at least about 15% or 25% in a standard in vitro cell proliferation assay. Examples of such an assay are described below. It is generally preferred that the administered compound exhibits an EC50 of at least about 10 xcexcM in a standard in vitro GalT-2 assay as defined below, more preferably an EC50 of about 1 xcexcM or less, still more preferably an EC50 of about 0.001 xcexcM or less in a standard in vitro GalT-2 assay as defined below. As defined herein, the EC50 is a concentration of the cell proliferation enhancing compound that exhibits at least about 10% to 20% stimulation of cell proliferation with respect to a suitable control as described below. Such compounds that can enhance GalT-2 activity are generally referred to herein as xe2x80x9cGalT-2 enhancing compoundsxe2x80x9d or other similar term.
Compounds suitable for use in the treatment methods of the invention are generally levorotatory and include those of the following Formula I: 
wherein R, R1, R2 and R3 are as defined below; and pharmaceutically acceptable salts of such compounds.
The term xe2x80x9clevortatoryxe2x80x9d (as opposed to xe2x80x9cdextrorotatoryxe2x80x9d) is used herein to denote compounds of Formula I that have capacity to rotate polarized light counterclockwise, ie. in the L or (xe2x88x92) direction as specifically defined below. Preferred Formula I compounds exhibit a specific optical rotation of at least xe2x88x925, 10, xe2x88x9220, xe2x88x9230, xe2x88x9250, xe2x88x92100, xe2x88x92200 up to about xe2x88x92300 degrees relative to a suitable optically inactive compound.
Specifically preferred enhancing compounds for use in the therapeutic methods of the invention include L-enantiomers of the following compounds:
1-phenyl-2-decanoylamino-3-morpholino-1-propanol;
1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol;
1-phenyl-2-hexadecanoylamino-3-piperidino-1-propanol;
1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol;
1-morpholino-2-hexadecanoylamino-3-hydroxyoctadec-4,5-ene; and
1-pyrrolidino-2-hexadecanoylamino-3-hydroxyoctadec-4,5-ene.
Especially preferred inhibitor compounds for use in the methods of the invention are L-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (L-PDMP) and trans-1-pyrrolidino-2-hexadecanoylamino-3-hydroxyoctadec-4,5-ene.
Other suitable GalT-2 enhancing compounds can be readily identified by simple testing, e.g. by in vitro testing of a candidate GalT-2 enhancing compound relative to a control for the ability to promote GalT-2 activity, e.g. by at least 10% more than a control.
The invention further relates to methods of detecting and analyzing compounds that promote GalT-2 activity and exhibit therapeutic capacity to treat or prevent the above-described conditions. Preferred detection and analysis methods include both in vitro and in vivo assays to determine the therapeutic capacity of agents to modulate LacCer-responsive cells.
Preferred in vitro detection assays according to the present invention involve one or more steps associated with LacCer-related pathways. Such assays include the following steps 1) through 4):
1) culturing a population of LacCer-responsive cells with LacCer;
2) adding a known or candidate GalT-2 enhancing compound to the cells;
3) measuring activity of a cell molecule or function in the LacCer-related pathway; and
4) determining the effect of the known or candidate GalT-2 enhancing compound on the cell, e.g., by measuring activity of the cell molecule or function. Typically, the cell function will be one or more of cell proliferation, cell adhesion or expression of specified surface proteins on the cells. Examples of cell molecules include LacCer-responsive enzymes as specified below.
That assay can effectively measure the capacity of the GalT-2 enhancing compound to promote GalT-2 activity. References herein to a xe2x80x9cstandard in vitro GalT-2 assayxe2x80x9d or other similar phrase refers to the above protocol of steps 1) through 4) when the specified cell molecule measured in step 3) above is GalT-2. As described in more detail below, other in vitro assays of the invention measure specified cell molecules in the LacCer-related steps or pathways. The in vitro assays of the present invention can be conducted with nearly any population of cells responsive to LacCer as provided below.
Suitable LacCer responsive cells that may be used or tested for compatibility with the standard in vitro GalT-2 assay include cells associated with vascular intima, particularly endothelial and smooth-muscle cells; kidney cells, neurons, glia as well as certain immune cells such as leukocytes. The cells may be immortalized, cultured or may be primary cells as desired. Additionally, tissue slices or organs may also be used.
Although it is generally preferred that whole cells be used in the assay, in some instances a lysate of such cells or tissue, or a substantially purified fraction of the lysate may be employed. Preferred LacCer lysates or subcellular fractions include GalT-2.
The in vitro detection assays of the invention can be adapted in accordance with intended use. For example, as noted above, it has been found that LacCer manifests changes in certain cell functions such as cell proliferation and cell adhesion. Thus, the standard in vitro GalT-2 assay above can be modified, (e.g., at step 3) to include measuring an increase in cell proliferation or adhesion (or both) in response to the added LacCer, and to determine any effect of the GalT-2 enhancing compound on the cell function. The known or candidate GalT-2 enhancing compound tested in the assay can be employed as a sole active agent or in combination with other agents including other GalT-2 enhancing compounds to be tested. In most instances, the in vitro assays are performed with a suitable control assay usually comprising the same test conditions as in the steps above, but without adding the GalT-2 enhancing compound to the medium. In such cases, a candidate GalT-2 enhancing compound can be identified as exhibiting desired activity by exhibiting at least about 10 percent greater activity relative to the control; more preferably at least about 20% greater activity relative to the control assay; and still more preferably at least about 30%, 40%, 50%, 60%, 70, 80%, 100%, 150% or 200% greater activity or more relative to the control.
The invention also provides assays to detect a LacCer-responsive cell which cells may be used, e.g., in an assay of the invention as described herein. In one assay, a potentially LacCer-responsive cell can be contacted by LacCer and then a desired cell molecule or function can be measured as a function of the amount of LacCer added. In most cases, the cell is deemed responsive to LacCer if the assay employed shows at least about 10%, preferably at least about 20%, more preferably at least about 50%, and still more preferably at least about 75% or 100% change in the activity of the molecule or cell function (relative to a control) as determined by the assays provided herein. The assays can be used to identify LacCer-responsiveness in a variety of cells or tissues, including cultured cells (i.e., primary cells or immortalized cell lines), tissue slices and organs.
The in vitro assays are particularly useful for detecting potential synergistic effects between a known or candidate GalT-2 enhancing compound and one or more other molecules, e.g, other GalT-2 enhancing compounds that can increase cell proliferation or adhesion. Examples of such potential molecules include growth factors, cytokines, polypeptides, peptides and particularly peptide hormones, and small molecules such as cyclic nucleotides and certain nucleosides.
The invention also provides in vivo assays to determine the therapeutic capacity of a known or candidate GalT-2 enhancing compound to modulate cell functions impacted by LacCer, e.g. cell proliferation, cell adhesion or both. The monitored cell function suitably may be pre-existing in the test animal, or the cell function may be induced, e.g., by administering a drug capable of modulating the cell function or by conducting an invasive surgical procedure such as angioplasty. In addition to cell proliferation and adhesion, cell functions that can be suitably assayed include, e.g., vessel remodeling, angiogenesis, regeneration of cells and tissue including particularly, tissue repair, and immune responses, e.g., recruitment of specified immune cells such as B and T cells.
Further suitable in vivo assays include those designed to evaluate overall neurological function in a test animal according to conventional methods. For example, the therapeutic capacity of a desired, known, or GalT- enhancing candidate compound can be tested by evaluating CNS and/or PNS function in the test animal. Such tests are known in the field and include those tests that are capable of measuring perception, cognition, motor skills (e.g., reflexes) and vision.
The in vivo assays of the present invention can be modified in a number of ways as needed. For example, in certain embodiments of the present invention relating to measuring vessel cell proliferation, a vessel subjected to analysis can be assayed in vitro following removal from the animal or assayed in vivo if desired. In most embodiments, activity of the GalT-2 enhancing compound in a given in vivo assay is compared to a suitable control (e.g., a sham-operated animal) in which the assay is conducted the same as the test assay but without administering the GalT-2 enhancing compound to the test subject. A variety of test subjects can be employed, particularly mammals such as rabbits, primates, various rodents and the like.
As noted above, the detection assays (either in vitro or in vivo) can be conducted in a wide variety of LacCer-responsive cells, tissues and organs. Further, the assays can detect useful GalT-2 enhancing compounds by measuring the activity of target molecules and/or functions related to the LacCer-related pathways. Thus, the present assays can measure activity in several cell, tissue and organ settings.
Significantly, use of multiple detection assays (e.g., a combination of the in vitro and/or in vivo assays) with a single GalT-2 enhancing compound can extend the selectivity and sensitivity of detection as desired.
Such broad spectrum testing provides additional advantages. Thus, for example, in vitro assays of the invention can efficiently perform multiple analyses, thereby increasing efficiency and probability of identifying GalT-2 enhancing compounds with therapeutic capacity. This is especially useful when large numbers of compounds need to be tested. For instance, libraries of GalT-2 enhancing compounds can be made by standard synthetic methods including combinatorial-type chemistry manipulations and then tested in accord with the invention.
Additionally, many of the LacCer-related steps are xe2x80x9cdownstreamxe2x80x9d of GalT-2, and therefore the assays include molecules and cell functions that are active downstream of GalT-2. Accordingly, modest but significant changes in GalT-2 activity can be registered as readily testable signals.
Other aspects of the invention are discussed below.